Structural domains influencing sensitivity to isothiourea derivative inhibitor KB-R7943 in cardiac Na+/Ca2+ exchanger

KB-R7943 (2-[2-[4-( 4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate) is a potent and selective Na+/Ca2+ exchange (NCX) inhibitor that is 3-fold more inhibitory to NCX3 than to NCX1 or NCX2. Here we searched for amino acid residues that may form the KB-R7943 receptor in the exchanger by analyzing the function of chimeras between NCX1 and NCX3 as well as of their site-directed mutants. We found that the highly conserved alpha -2 repeat of the exchanger is almost exclusively responsible for the difference in drug response of the isoforms. Such difference was mostly reproduced by single substitutions of residues in the alpha -2 repeat (V820G or Q826V in NCX1 and A809V or A809I in NCX3), suggesting their importance in drug sensitivity. Cysteine scanning mutagenesis of the alpha -2 repeat of NCX1 identified one residue (Gly833) that caused a large (greater than or equal to 30- fold) reduction in drug sensitivity. We found that the Gly-to-Thr substitution caused even larger reduction in drug sensitivity. Interestingly, extracellularly applied KB-R7943 at 0.8 muM markedly inhibited the whole-cell outward exchange current, whereas the drug applied intracellularly at 30 muM did not. These results suggest that KB-R7943 inhibits the exchanger from the external side in intact cells and that a region of the alpha -2 repeat of NCX1 containing Gly833 may participate in the formation of the drug receptor. Because we suggested previously that Gly833 is accessible from the inside of a cell, the results raised an interesting possibility that this residue may alter its position during Na+/Ca2+ exchange in such a way that it becomes accessible to external drug.